Cancers of the esophagogastric region are highly malignant tumours with five-year survival rates of less than sixteen percent (Sant et al., 2003). Research has shown that 88% of patients, selected for curative resection for esophagogastric cancer, already have disseminated tumour cells (O'Sullivan G et al., 1999), that can remain dormant for variable periods, before emerging as aggressive, drug resistant metastases (Ryan et al., 2004). Improved systemic therapeutic options are therefore required to effectively eliminate primary and recurrent esophageal cancer.
Chemotherapeutic regimes are designed to induce maximum cancer cell killing, by engaging a cell death program. Drug resistance due to a failure to adequately engage programmed cell death (PCD) leads to recurrence of cancer. This is a major limitation, as de-regulation of cell death programs often plays a role in the development of the cancer in the first place (Raguz and Yague, 2008). Previously, apoptosis (Type I cell death) was regarded as the central mediator of PCD in response to chemotherapeutic agents. However, other death programs exist in eukaryotic cells (Ricci and Zong, 2006, Degterev and Yuan, 2008). Type II cell death is characterised by the formation of vesicles in the cytoplasm, loss of the cytoplasmic material and pyknosis of nuclear material within an intact nuclear membrane (Clarke, 1990). Evidence suggests that this morphology is a consequence of excessive autophagy. Several studies have now reported autophagic cell death in cultured mammalian cells (Pattingre et al., 2005, Yu et al., 2006, Opipari et al., 2004, Scarlatti et al., 2008, Debnath et al., 2005). Furthermore, autophagic programmed cell death has now been demonstrated during development of Drosophila and Dictyostelium discoideum (Berry and Baehrecke, 2007, Lam et al., 2008).
Autophagy is a highly conserved survival response to growth limiting conditions, in which cellular components are sequestered, degraded and released for re-cycling by autophagosomes (Yorimitsu and Klionsky, 2005). It is genetically regulated by a family of Atg genes (Mizushima, 2007) which have homologues in humans (e.g. human ortholog of Atg6-Beclin1). The role of autophagy in cancer remains controversial. Constitutive autophagy may be a necessary homeostatic process which removes damaged organelles and re-cycles macromolecules thus protecting against cancer (Mizushima et al., 2008). However, when a cancer is established—autophagy may take on new roles—it may help cancer cells survive in response to growth limiting conditions such as nutrient depletion, hypoxia, absence of growth factor and presence of cytotoxic drug (Jin and White, 2008, Degenhardt et al., 2006, Amaravadi et al., 2007). The induction of excessive autophagy may also be the major cell death mechanism that takes over when apoptosis is unavailable (Scarlatti et al., 2009). Autophagic cell death has been reported to be induced in malignant gliomas, ovarian and breast carcinoma by the chemo-therapeutic agents temozolomide and Tamoxifen (Kanzawa et al., 2003, Kanzawa et al., 2004, Takeuchi et al., 2005, Opipari et al., 2004).
It is an object of the invention to overcome at least one of the above-referenced problems.
Statements of the Invention
The invention is based on the surprising finding that treatment with a chemotrerapeutic agent such as 5-fluorouracil (5-FU) and an autophagy inducer effectively inhibit the continued growth of, and prevent the recovery following drug withdrawal, of cancer cells. In vivo, drug resistance from a failure to adequately engage in apoptotic programmed cell death leads to a recurrence of cancer and tumours can remain dormant for periods of time before re-emerging as drug resistant metastases. It has been hypothesised that autophagy (Type II cell death) may help cancer cells survive in response to growth limiting conditions, such as nutrient depletion, hypoxia, absence of growth factor, or presence of cytotoxic drug. LiCl is a known autophagy inducer and accelerates cell survival to autophagic programmed cell death. The Applicant has shown that the combination of an autophagy inducer and a chemotherapeutic agent prevented the recovery of apoptosis competent and apoptosis incompetent cancer cells. The Applicant has also shown in an in vivo cancer model that the combination of an autophagy inducer and chemotherapeutic agent stops tumour growth and in fact reduces tumour volume to such an extent that the tumour disappears and does not return following cessation of treatment.
Accordingly, the invention broadly relates to a method of treatment and/or prevention of cancer in an individual comprising a step of administering to the individual a therapeutically effective amount of at least one chemotherapeutic agent and at least one autophagy inducer.
Suitably, the invention relates to a method for the treatment and/or prevention of a chemo-resistant cancer in an individual comprising a step of administering to the individual a therapeutically effective amount of at least one chemotherapeutic agent and at least one autophagy inducer.
In a preferred embodiment, the invention provides a method of treating an epithelial cancer, typically selected from lung, breast, colorectal and esophagogastric cancer, especially esophageal cancer, comprising administering to an individual in need thereof a therapeutically effective amount of at least one chemotherapeutic agent and at least one autophagy inducer.
Suitably, the chemotherapeutic agent is selected from a pyrimidine analogue (for example 5-FU) and a DNA-binding heavy metal ion complex such as platinum, palladium, ruthenium or osmium complex. In one embodiment, at least two chemotherapeutic agents are employed, for example 5-FU and a DNA-binding heavy metal ion complex.
Thus, in one embodiment, the methods of the invention comprise administering at least one authophgy inducer with at least two chemotherapeutic agents, for example 5-FU and oxaliplatin as chemotherapeutic agents and LiCl or an alternative autophagy inducer (for example rapamycin or a rapamycin derivative such as everolimus).
The invention also relates to a method of preventing recovery of cancer cells upon withdrawal of a chemotherapeutic agent, the method comprising a step of treating the cancer cells with an autophagy inducer. The cells may be treated with the autophagy inducer at the same time as they are treated with the chemotherapeutic agent, and/or they may be treated after the chemotherapeutic treatment has been withdrawn.
The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a chemotherapeutic agent and a therapeutically effective amount of an autophagy inducer. Typically, the ratio of autophagy inducer to chemotherapeutic agent is from 50:1 to 1:1, suitably from 20:1 to 2:1 (mg/kg body weight).
In another embodiment, the invention relates to the use of a chemotherapeutic agent and an autophagy inducer in the manufacture of a medicament for the treatment and or prevention of cancer.
The invention also relates to a pharmaceutical kit comprising an amount of a chemotherapeutic agent and an amount of an autophagy inducer.
Suitably, the composition or kit comprises one or more chemotherapeutic agents selected from 5-FU, and DNA-binding heavy metal ion complex (such as for example platinum complexes). In one embodiment, the composition or kit comprises at least one autophagy inducer, for example a lithium salt, and at least two chemotherapeutic agents (for example a pyrimidine analogue such as 5-FU and a DNA-binding heavy metal ion complex such as cisplatin, carboplatin or oxaliplatin).
Preferably, the pharmaceutical composition comprises:                a lithium salt and 5-FU, optionally in combination with a further chemotherapeutic agent;        a lithium salt and a platinum complex selected from oxaliplatin, carboplatin, and oxaliplatin, optionally in combination with a further chemotherapeutic agent;        a BH3 mimetic and 5-FU, optionally in combination with a further chemotherapeutic agent;        a BH3 mimetic and a platinum complex selected from oxaliplatin, carboplatin, and oxaliplatin, optionally in combination with a further chemotherapeutic agent;        rapamycin and 5-FU, optionally in combination with a further chemotherapeutic agent;        rapamycin and a platinum complex selected from oxaliplatin, carboplatin, and oxaliplatin, optionally in combination with a further chemotherapeutic agent;        everolimus and 5-FU, optionally in combination with a further chemotherapeutic agent; or        everolimus and a platinum complex selected from oxaliplatin, carboplatin, and oxaliplatin, optionally in combination with a further chemotherapeutic agent.        
In another embodiment, the invention relates of a method for preventing the recovery of cancer cells comprising the steps of treating the individual with a therapeutically effective amount of chemotherapeutic agent and a therapeutically effective amount of an autophagy inducer.
The invention also relates to a method of treating an individual with cancer and who is undergoing treatment with a chemotherapeutic agent, the method comprising the step of co-treating the individual with a therapeutically effective amount of autophagy inducer.
In another embodiment, the invention relates to a method for treating an individual with cancer, the method comprising the step of treating the individual with a therapeutically effective amount of an autophagy inducer.
Definitions
Typically, the cancer is selected from the group comprising: esophagogastric cancer; fibrosarcoma; myxosarcoma; liposarcoma; chondrosarcoma; osteogenic sarcoma; chordoma; angiosarcoma; endotheliosarcoma; lymphangiosarcoma; lymphangioendotheliosarcoma; synovioma; mesothelioma; Ewing's tumor; leiomyosarcoma; rhabdomyosarcoma; colon carcinoma; colorectal carcinoma; pancreatic cancer; breast cancer; ovarian cancer; prostate cancer; squamous cell carcinoma; basal cell carcinoma; adenocarcinoma; sweat gland carcinoma; sebaceous gland carcinoma; papillary carcinoma; papillary adenocarcinomas; cystadenocarcinoma; medullary carcinoma; bronchogenic carcinoma; renal cell carcinoma; hepatoma; bile duct carcinoma; choriocarcinoma; seminoma; embryonal carcinoma; Wilms' tumor; cervical cancer; uterine cancer; testicular tumor; lung carcinoma; small cell lung carcinoma; bladder carcinoma; epithelial carcinoma; glioma; astrocytoma; medulloblastoma; craniopharyngioma; ependymoma; pinealoma; hemangioblastoma; acoustic neuroma; oligodendroglioma; meningioma; melanoma; retinoblastoma; primary and metastatic tumors, and leukemias. Typically, treatment of the cancer entails reducing one or more of survival, proliferation and migration of, or invasion by, cancer cells.
In this specification, the term “chemo-resistant cancer” should be taken to mean cancer cells that exhibit autophagy following exposure to chemotherapeutic agents.
In this specification, the term “treatment” should be taken to mean a course of action/dosing regime that either inhibits, delays or prevents the progression of cancer, including cancer metastasis, or that inhibits, delays or prevents the recurrence of cancer, including cancer metastasis, or that prevents or hinders the onset or development of cancer in an individual.
In this specification, the term “prevention” should be taken to mean prevention of the recurrence of cancer, at a local or distant site, typically following the withdrawal of chemotherapeutic drugs in an individual diagnosed with cancer.
In this specification, the term “chemotherapeutic agent” should be taken to mean an agent that induces cancerous cells to commit to cell death. Suitable chemotherapeutic agents will be known to those skilled in the art. Such chemotherapeutic agents include but are not limited to; alkylating agents, anti-metabolites, plant alkyloids and terpenoids, topoisomerase inhibitors, anti-tumour antibiotics, DNA-binding heavy metal ion-based complexes including but not limited to the platinum-based complexes cisplatin, carboplatin and oxaliplatin, and histone deacetylase (HDAC) inhibitors including hydroxamate-type HDAC inhibitors (SAHA, Pabinostat, Belinostat) and benzamide-type HDAC inhibitors (the details of which will be well known to those skilled in the art. Examples of suitable chemotherapeutic anti-metabolites include, purine analogues not limited to azathoprine, mercaptopurine, tioguanine and fludarabine; pyrimidine analogues not limited to 5-fluorouracil (5-FU), floxuridine and cytosine arabinoside; antifolates not limited to methotrexate, trimethoprim, pyrimethamine and pemetrexed. Suitably, the chemotherapeutic agent is a DNA damaging agent (to include DNA-binding agent). Preferably, it is a pyrimidine analogue, examples of which are provided above. Ideally, it is 5-FU.
In this specification, the term “autophagy inducer” should be taken to mean an agent which induces cancer cells to commit to an autophagic process. Suitable inducers of autophagy will be well known to those skilled in the art. One example is a lithium compound, for example a lithium salt. Examples of lithium salts are lithium chloride (LiCl) or any other pharmaceutically acceptable salts thereof, including but not limited to; lithium carbonate, lithium citrate, lithium sulfate, lithium aspartate, lithium orotate. Another example of a class of compounds that induce autophagy are BH3 mimetics such as, for example, HA14-1 (Sigma Ireland). For a detailed review on how BH3 mimetics are proposed as a promising anticancer agent see (Zhang et al., 2007). Rapamycin (also known as sirolimus), and rapamycin analogues, for example everolimus, temsirolimus, are further examples of autophagy inducers, the details of which will be well known to those skilled in the art.
In the specification, the term “individual” should be taken to mean a human; however it should also include higher mammals for which the therapy of the invention is practicable.
In this specification, the term “therapeutically effective amount” should be taken to mean an amount of a chemotherapeutic agent and an autophagy inducer which result in partial or total inhibition in the progression of cancer and prevent or inhibits the recurrence of cancer following withdrawal from an anti-cancer regime. In a particular, a therapeutically effective amount of a chemotherapeutic agent should be taken to mean an amount that results in a clinically significant number of cancer cells being killed. A therapeutically effective amount of an autophagy inducer should be taken to mean an amount that results in a clinically significant number of chemoresistant cancer cells being killed by means of Type II cell death. An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors are considered by the attending diagnostician, including, but not limited to: the type of chemotherapeutic agent; species of mammal; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances. As an example, the following doses may be employed:    Cisplatin: high dose=6.9 mg/kg; low dose=2 mg/kg    Oxaliplatin: high dose=6/15 mg/kg; low dose=1.5/5 mg/kg;    Lithium Chloride: high dose=14.5/17 mg/kg; low dose=4.5/10 mg/kg    Rapamycin: high dose=2 mg/kg; low dose=0.6 mg/kg    5-Fluorouracil: high dose=87 mg/kg; low dose=8/12 mg/kg
In this specification, the term “administering” should be taken to include any form of delivery that is capable of delivering the chemotherapeutic agent and the autophagy inducer to cancer cells including local delivery, intravenous delivery, oral delivery, intramuscular delivery, intrathecal delivery, transdermal delivery, inhaled delivery and topical delivery. Methods for achieving these means of delivery will be well known to those skilled in the art of drug delivery. The term should also encompass co-administration of the two active compounds, or administration at separate times. For example, the actives may be administered on alternate days, or on the same day at different times, or on different days of the week.
In one preferred embodiment, the drugs are co-administered. One suitable way of achieving this is the provision of both drugs in a unit dose form, for example a pharmaceutical formulation comprising the two drugs in the form of a tablet or a capsule. In the unit dose, the drugs may be admixed, or they may be kept separate in different parts of the unit dose. For example, the unit dose may be a capsule having the drugs separated into different compartments of the capsule.
The chemotherapeutic agent and an autophagy inducer may form part of the same pharmaceutical composition or may comprise separate components for administration in a therapeutically effective amount at the same or different times and in any order or sequence.
In this specification, the term “pharmaceutical composition” should be taken to mean compositions comprising a therapeutically effective amount of a chemotherapeutic agent and an autophagy inducer, and a pharmaceutically acceptable carrier or diluent. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the chemotherapeutic agent and an autophagy inducer is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.
The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to, ease pain at the, site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.